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Abstract

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We found that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier – consistent with a sieve-like phase. Phase separation tolerated mutations of Nup98-typical GLFG motifs, provided the domain-hydrophobicity remained preserved. NTR-entry, however, was sensitive to (certain) deviations from canonical FG motifs, suggesting a co-evolutionary adaptation. Unexpectedly, we found that arginines promote efficient FG-phase entry also by means other than cation-π interactions. Although incompatible with NTR·cargo complexes, a YG phase displayed remarkable transport selectivity, particularly for evolved GFP^NTR-variants. GLFG to FSFG mutations made the FG phase hypercohesive, precluding NTR-entry. Longer spacers relieved this hypercohesive phenotype. The antagonism between cohesion and NTR·FG interactions appears thus key to transport selectivity.